1. Field of the Invention
The present invention relates to a time-of-flight secondary ion mass spectrometer which can acquire information on a sample using a time-of-flight mass spectrometric unit. More specifically, the present invention relates to a time-of-flight secondary ion mass spectrometer which can perform imaging detection efficiently every kind of compositions which construct a sample, and in particular, organic substances such as protein and peptide (hereinafter, “polypeptide”).
2. Description of the Related Art
A close-up of importance of analysis of protein which is a gene product which exists in a living body has been rapidly taken by development of genome (genome) analysis in recent years. In addition, up to now, importance of expression and performance analysis of protein has been pointed out, and development of their analytic methods has been advanced. These methods are based on combination of (1) separation refinement by two-dimensional electrophoresis or a High Performance Liquid Chromatograph (HPLC), and (2) a detection system such as radiometric analysis, optical analysis or mass analysis.
A ground of this protein analysis technique is called proteome (proteome) analysis, and this analyzes protein which is made from a gene and is actually acting in a living body. Then, it aims at finally investigating functions of a cell, and a cause of a disease. The following methods can be cited as typical analytic methods of this proteome analysis.
(1) Extraction of protein from a living body tissue or a cell which is a target
(2) Separation of protein by two-dimensional electrophoresis
(3) Analysis of protein or its fragment by mass analysis such as a MALDI method (Matrix Support Laser Desorption-Time-Of-Flight Mass Spectrometry: MALDI-TOFMS)
(4) Proteinic identification using databases such as the Genome Project database
On the other hand, the present inventor proposed an information acquisition method and apparatus, which use the TOF-SIMS method (time-of-flight secondary ion mass spectrometry) as a base, in Japanese Patent Application Laid-Open No. 2006-10658. These information acquisition method and apparatus aim at visualization of a two-dimensional distribution of polypeptide in a protein chip or a cut piece of a living body tissue. This method attaches an ionization promoting agent and a digestive enzyme to the above-mentioned protein chip and cut piece of a living body tissue using an ink jet method or the like. Then, this method visualizes information (including information on peptide which is limitedly decomposed with the digestive enzyme) regarding a kind of protein by the TOF-SIMS method with keeping positional information.
Furthermore, as an example of analyzing polypeptide by the TOF-SIMS method, a method of detecting a polypeptide parent molecule with a large molecular weight by performing the same pretreatment as the MALDI method, that is, mixing polypeptide with a matrix substance is disclosed in A. F. Maarten et al. Anal. Chem., vol. 77, 735 (2005).
In D. G. Castner, Nature 422, and 129 (2003), a method of promoting ionization of high polymers with suppressing fragmentation, and improving ion detection sensitivity as a result is disclosed.
In the document, as a method of analyzing polypeptide effectively, examples using carbon 60 Fullerene (C60) ions, trimer cluster (Au3, Bi3) ions of metallic elements such as gold and bismuth and the like as primary ions in the TOF-SIMS method are described. Thereby, energy multiple scattering in very shallow bounds of a sample surface arises by radiation of primary ions. Then, it is described that many polymers which exist near the surface where the primary ions collide can be emitted (sputtering) softly.
In both of documents of Japanese Patent Application Laid-Open No. H07-211282 and Japanese Patent Application Laid-Open No. 2005-300480, methods of promoting fragment ionization of high polymers by improvement of an ionization mechanism in an analytical instrument including a time-of-flight mass spectrometer other than the TOF-SIMS method to obtain information on molecular structure are disclosed. More specifically, the former radiates a collision gas between an ion source and a mass spectrometric unit, and the latter makes an infrared laser beam radiated between an ion source and a mass spectrometric unit.
By an information acquisition method described in the above-mentioned Japanese Patent Application Laid-Open No. 2006-10658, information (including information on peptide which is limitedly decomposed by a digestive enzyme) regarding protein of a diseased tissue and a normal tissue is acquirable. Nevertheless, depending on a kind and a measuring condition of a sample, there was a case where detection sensitivity was not sufficient.
On the other hand, the method of Maarten et al. is a method which can suppress decomposition by primary ion irradiation even if it is polypeptide with a large molecular weight, and can detect a parent molecule with keeping original mass. Nevertheless, since this method made a test sample what polypeptide and a matrix substance are mixed, when a sample such as a protein chip is analyzed, it is not able to acquire original two-dimensional distribution information.
As improvement methods of apparatuses which use the TOF-SIMS method and solve the above issues, it is conceivable to apply methods, such as a MALDI method and an LC-TOFMS (Liquid Chromatograph Time-Of-Flight Mass Spectrometer) method. What is conceivable as its typical example is a method which is described in Japanese Patent Application Laid-Open No. H07-211282 and Japanese Patent Application Laid-Open No. 2005-300480 which are cited previously, and radiates a collision gas or an infrared laser beam between an ion source and a mass spectrometric unit to perform fragment ionization of and to detect polypeptide high polymers. Nevertheless, since a sample used as a measuring object was limited in these methods, and there were also few secondary ion amounts of emergence from a sample since the collision gas or infrared laser beam had comparatively low energy, measurement accuracy was limited.
Recently, an improved method widely used as a realistic method is the previously cited Castner's method of using metallic cluster ions such as C60, Au3, and Bi3 for primary ions. This method induces multiple scattering in a very shallow region of a sample surface by radiating metallic cluster ions as primary ions on a sample, and can emit polymer ions (including neutral ions) favorably with suppressing fragmentation. This method has an advantage of being possible not only to increase detection sensitivity of a polymer ion, but also to detect position distribution information in submicron order which is a characteristic of a metal ion beam. That is, this method has an advantage of improving the detection sensitivity of a polypeptide ion with a mass number of 200 to 1000 up to tens of times to hundreds of times in comparison with a case of gallium or argon which was conventionally used as a primary ion source.
Nevertheless, the Castner's method performs measurement and reforming of a sample surface by primary ion irradiation simultaneously. For this reason, an emission efficiency of the polymer ions from a sample was poor. In addition, since this method emitted polymer ions with inducing multiple scattering broadly, regions where these primary metallic cluster ions were radiated on a sample surface were extremely little to the extent of one atom per 100 atoms. For this reason, there was a problem that many portions of the sample surface were consumed vainly without providing an analysis.
As described above, it was hard in the analysis method using a conventional TOF-SIMS method to detect parent molecule ions of polypeptide with high sensitivity with maintaining positional information to a sample such as a protein chip or an organism specimen.